Purification of green fluorescent protein from a crude solution has been investigated using a combined system of ion-exchange chromatography and size-exclusion chromatography packed into one column of a continuous annular chromatograph. Performing the chromatography with two different sorbents packed into a single column reduces transfer losses and the hold steps between the two separation steps can be eliminated. Appropriate running conditions for the continuous separation mode were achieved by searching for optimal ones in conventional batch-wise operation. Green fluorescent protein was expressed in Saccharomyces cerevisiae, enzymatically lysed and the extract was used as the feed stock. Superdex 200 prep grade was packed into an annular chromatograph (outer diameter: 15 cm, inner diameter: 14 cm) and on top a 1.5 cm layer of the anion-exchange sorbent Source 30 Q. Green fluorescent protein was enriched by the anion-exchange resin and further purified by the size-exclusion gel. Purity was analyzed by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, fluorescence intensity, and analytical size-exclusion chromatography.